1. Field of the Invention
The invention relates to materials and methods for preparing vaccines, and more particularly to genetically engineered microorganisms which are useful to express desired gene products in the immunized animal host because they are balanced lethals which can be maintained as a genetically stable population within the immunized animal host.
2. Description of the Related Art
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Genetically engineered microorganisms have widespread utility and importance. One important use of genetically engineered microorganisms is as a live vaccine for inducing immunity. See, e.g., U.S. Pat. Nos. 6,024,961; 4,888,170; 5,389,368; 5,855,879; 5,855,880; 5,294,441; 5,468,485; 5,387,744; 5,840,483, 5,672,345; 5,424,065; 5,888,799; 5,424,065; 5,656,488; 5,006,335; 5,643,771; 5,980,907; 5,851,519; and 5,527,529, all of which are incorporated by reference. When the genetically engineered microorganism is to be utilized as a vertebrate live vaccine, certain considerations must be taken into account. To provide a benefit beyond that of a nonliving vaccine, the live vaccine microorganism must attach to, invade, and survive in lymphoid tissues of the vertebrate and expose these immune effector sites in the vertebrate to antigen for an extended period of time. By this continual stimulation, the vertebrate's immune system becomes more highly reactive to the antigen than with a nonliving vaccine. Therefore, preferred live vaccines are attenuated pathogens of the vertebrate, particularly pathogens that colonize the gut-associated lymphoid tissue (GALT) or bronchial-associated lymphoid tissue (BALT). An additional advantage of these attenuated pathogens over nonliving vaccines is that these pathogens have elaborate mechanisms to gain access to lymphoid tissues, and thus efficient exposure to the vertebrate's immune system can be expected. In contrast, nonliving vaccines will only provide an immune stimulus if the vaccine is passively exposed to the immune system, or if host mechanisms bring the vaccine to the immune system.
Despite their advantages over non-living vaccines, effective live vaccines must ovecome certain obstacles. Genetically engineered microorganisms used as vaccines for antigen delivery must synthesize a gene product from which it derives no benefit, and the high level expression of the recombinant protein may be deleterious to the microorganism. Thus, the genetically engineered microorganism may be at a selective disadvantage relative to the same type of microorganism that does not produce the cloned gene product. As a result, when the vaccine is being manufactured, e.g., in a fermentor during production of the vaccine, spontaneous segregants that have lost the DNA sequence specifying the desired gene product quickly outpopulate the genetically engineered microorganism. This loss of the antigen-producing DNA sequence can also occur to the vaccine after inoculation into the host animal. Therefore, selection mechanisms have been developed which are designed to maintain the antigen-producing DNA sequence in the microorganism population.
One method for applying selective pressure to a bacterial population to maintain production of the desired polypeptide is to insert the recombinant gene encoding the polypeptide in a plasmid that also contains a gene encoding antibiotic resistance. Most cloning vectors currently in use have one or more genes specifying resistance to antibiotics. Thus, antibiotics can be added to the culture medium for growth of genetically engineered microorganisms to kill those bacteria that have lost the recombinant plasmid. This practice has several drawbacks. First, it is expensive to add antibiotics to growth medium. Second, since antibiotic resistance is often based upon the synthesis of drug inactivating enzymes, cells remain phenotypically drug resistant for a number of cell generations after the loss of genes for drug resistance and the linked desired gene. Third, in the case of genetically engineered bacteria to be used as a live vaccine, the United States Department of Agriculture and the Food and Drug Administration have refrained from approving strains which express antibiotic resistance.
An alternative to the use of antibiotic resistance for maintaining a recombinant plasmid and/or a cloned gene in a genetically-engineered microorganism is the use of a mutant microorganism that lacks a critical biosynthetic enzyme, and supplying the wild-type gene for that enzyme on the plasmid cloning vector. See, e.g., Kahn et al (1979) and Dean (1981). Unfortunately, this is impractical in many situations. The use of mutants which are missing enzymes involved in the biosynthesis of amino acids, purines, pyrimidines, and vitamins often does not preclude the growth of these mutants since the end-product of the pathway which is required for growth is often furnished by the environment. For example, inexpensive media used for the growth of recombinant organisms in fermenters often contain these end products. In addition, particularly in the case of live vaccines, the end product may be supplied in vivo by the vaccinated host.
The problems of genetic instability of genetically engineered microorganisms possessing a cloned gene on a plasmid can arguably be alleviated by integrating the cloned gene into the chromosome of the microorganism. However, integration of the recombinant gene into the chromosome overcomes many of the potential benefits of having it reside on the plasmid. For example, control of plasmid copy number by, for example, selection of the plasmid containing the cloned gene provides a mechanism for increasing the product yield. It is to be noted that the level of expression of a gene is usually proportional to gene copy number, which increases with increasing plasmid copy number. The use of plasmids with a regulatable promoter also offer one mechanism for temporally controlling the expression of the product so that high level expression occurs at less deleterious times during the growth cycle.
All bacteria have a peptidoglycan layer of the cell wall that imparts shape and rigidity. The peptidoglycan is made of a polymer of repeating muramic acid-N-acetylglucosamine units and is cross-linked by short peptides. In all Gram-negative bacteria and in Mycobacterium and in Nocardia species of Eubacteria, the peptide is composed of L-alanine, D-glutamic acid, mesodiaminopimelic acid (DAP), and D-alanine. In most Gram-positive microorganisms the DAP component is replaced by its decarboxylation product L-lysine.
As illustrated by FIG. 1, DAP is synthesized in six enzymatic steps from β-aspartic semialdehyde, which, in turn, is synthesized in two steps from L-aspartic acid. In the first step, L-aspartic acid is phosphorylated by one of several (usually three) β-aspartokinases which are encoded by several (usually three) separate genes regulated independently by repression and/or feedback inhibition of the gene products by the ultimate end products L-threonine, L-methionine, and L-lysine. β-aspartyl phosphate is converted in one step to β-aspartic semialdehyde by β-aspartate semialdehyde dehydrogenase, the product of the asd gene. Mutants with a point mutation in or deletion of the asd gene as well as mutants with mutations in any of the six genes specifying the enzymes for converting β-aspartate semialdehyde to DAP require DAP in all media. When DAP-requiring mutants are deprived of DAP they undergo DAP-less death and lysis, releasing their contents.
The inclusion of asd, and thus dap, mutations in strains of bacteria affords biological containment, since such mutant strains are unable to survive in environments other than a carefully controlled laboratory environment. The basis for this has been extensively described in U.S. Pat. No. 4,190,495.
The gene for β-aspartate semialdehyde dehydrogenase from Streptococcus mutans PS14 (UAB62) has been cloned and expressed in asd mutants of E. coli (Jagusztyn-Krynicka, et al, 1982; Curtiss et al, 1982). Subsequently, the S. mutans asd gene was sequenced (Cardineau and Curtiss, 1987). The gene for β-aspartate semialdehyde dehydrogenase from Salmonella typhimurium has also been cloned and expressed in asd mutants of E. coli (Galán et al., 1990). Subsequently, the S. typhimurium asd gene was sequenced (SEQ ID NO:1) and its amino acid sequence determined (SEQ ID NO:2). Both sequences are found in Genbank accession number AF 015781.
U.S. Pat. No. 5,672,345 discloses a method of maintaining a desired recombinant gene in a genetic population of bacterial cells expressing the product of the desired recombinant gene. The method utilizes host cells having a mutation in a chromosomal gene encoding an enzyme that catalyzes a step in the biosynthesis of an essential cell wall component such as DAP. The host cells are transformed with two recombinant genes in physical linkage: one gene encoding a polypeptide that functionally replaces the enzyme and the other gene encoding the desired gene product. Loss of the recombinant gene complementing the mutant host gene causes the bacterial cells to lyse when in an environment requiring expression of the enzyme. The specification of patent 5,672,345 teaches that it is preferable that the non-functional chromosomal gene lack homology with its complementing plasmid gene, or have an extensive enough mutation (e.g., by utilizing a deletion mutation which eliminates the entire gene and/or flanking sequences) to preclude the ability of the complementing plasmid gene from recombining to replace the defective chromosomal gene by two crossover events on either side of the defective chromosomal gene. Such a lack of recombination maximizes the stability of the gene encoding the desired gene product by maintaining the linked selective pressure with the complementing recombinant plasmid gene.
However, avoiding homology between both sides of the inactivating chromosomal gene mutation and its functional plasmid counterpart precludes the use of many useful combinations of these genes. Therefore, it would be desirable to be able to utilize mutant chromosomal genes with complementing plasmid genes having such homology.